Human papillomaviruses (HPV), especially genotypes 16 and 18, are probable effectors of human urogenital malignancies. Although the male urethra is a proposed reservoir of HPV transmission, the association between HPV and squamous cell carcinoma (SCC) of the male urethra has not been studied. The highly sensitive technique of polymerase chain reaction with type-specific HPV 16 and 18 primers and general primers, including nine other genotypes was used to survey a series of SCC of the male urethra for the prevalence of an association with HPV. Archival surgical specimens from 14 patients were analyzed, and primary, recurrent, and metastatic lesions from 4 (29%) patients contained HPV 16 DNA. No other HPV genotype (6b, 11, 13, 18, 30, 31, 33, 35, 45, 51) was detected. Complete concordance for the presence of HPV in primary and recurrent or metastatic disease was demonstrated. These findings strongly suggest that HPV type 16 is associated with a substantial subset of SCCs of the male urethra. Analysis of clinical data revealed that HPV-positive tumors had a significant predilection for location in the pendulous urethra versus the bulbar urethra. Survival data analysis showed that the presence of HPV more closely correlated with prolonged survival than did tumor location. The presence or absence of HPV 16 DNA defines two subsets of SCC of the male urethra which differ in the site of occurrence and, possibly, progression.